Caspase-3 Research Articles

Effects of Pomegranate Seed Oil on Lower Extremity Ischemia-Reperfusion Damage: Insights into Oxidative Stress, Inflammation, and Cell Death

Aim: This study sought to clarify the therapeutic benefits and mechanisms of action of pomegranate seed oil (PSO) in instances of ischemia–reperfusion (IR) damage in the lower extremities. Materials and Methods: The sample size was determined, then 32 rats were randomly allocated to four groups: Control (C), ischemia–reperfusion (IR), low-dose PSO (IR + LD, 0.15 mL/kg), and high-dose PSO (IR + HD, 0.30 mL/kg). The ischemia model in the IR group was established by occluding the infrarenal aorta for 120 min. Prior to reperfusion, PSO was delivered to the IR + LD and IR + HD groups at doses of 0.15 mL/kg and 0.30 mL/kg, respectively, followed by a 120 min reperfusion period. Subsequently, blood and tissue specimens were obtained. Statistical investigation was executed utilizing Statistical Package for the Social Sciences version 20.0 (SPSS, IBM Corp., Armonk, NY, USA). Results: Biochemical tests revealed significant variations in total antioxidant level (TAS), total oxidant level (TOS), and the oxidative stress index (OSI) across the groups (p < 0.0001). The IR group had elevated TOS and OSI levels, whereas PSO therapy resulted in a reduction in these values (p < 0.05). As opposed to the IR group, TASs were higher in the PSO-treated groups. Histopathological analysis demonstrated muscle fiber degeneration, interstitial edema, and the infiltration of cells associated with inflammation in the IR group, with analogous results noted in the PSO treatment groups. Immunohistochemical analysis revealed that the expressions of Tumor Necrosis Factor-alpha (TNF-α), Nuclear Factor kappa B (NF-κB), cytochrome C (CYT C), and caspase 3 (CASP3) were elevated in the IR group, while PSO treatment diminished these markers and attenuated inflammation and apoptosis (p < 0.05). The findings demonstrate that PSO has a dose-dependent impact on IR injury. Discussion: This research indicates that PSO has significant protective benefits against IR injury in the lower extremities. PSO mitigated tissue damage and maintained mitochondrial integrity by addressing oxidative stress, inflammation, and apoptotic pathways. Particularly, high-dose PSO yielded more substantial enhancements in these processes and exhibited outcomes most comparable to the control group in biochemical, histological, and immunohistochemical investigations. These findings underscore the potential of PSO as an efficacious natural treatment agent for IR injury. Nevertheless, additional research is required to articulate this definitively.

Effects of Nicotinamide Riboside Supplementation on Postmortem Mitochondrial Functionality and Apoptotic Activation.

Early postmortem mitochondrial function and apoptotic activation affect meat quality development. Nicotinamide riboside (NR) supplementation to pigs prior to harvest can improve pork color stability, but its mechanism remains unclear. This study aimed to evaluate the impact of NR supplementation on early postmortem mitochondrial functionality and apoptosis. Sixteen pigs (N = 16) were individually fed a control or NR-supplemented diet (30 mg·kg body weight-1·d-1) for 10 days prior to harvest. Longissimus dorsi muscle samples were collected at 45 min and 24 h postmortem and analyzed for mitochondrial functionality using high-resolution respirometry and apoptotic protein abundance (apoptosis regulator Bcl-2-associated X (BAX), apoptotic inducing factor (AIF), and caspase 3 (CASP3)) via immunoblotting. NR-supplemented muscle exhibited lower proton leak-associated respiration at 45 min postmortem (p < 0.05), followed by a slower accumulation of mitochondrial outer membrane permeabilization (MOMP; p < 0.05) and a slower loss of mitochondrial integral function (p < 0.05) from 45 min to 24 h postmortem. NR supplementation decreased BAX abundance at 45 min postmortem but increased mature AIF abundance (62 kDa) at 24 h postmortem (p < 0.05). The abundance of CASP3 fragments (~29 kDa) decreased from 45 min to 24 h postmortem, independent of treatment (p < 0.05). NR supplementation demonstrated the potential to protect mitochondrial integral function and alleviate apoptotic activation in early postmortem porcine skeletal muscle, which might contribute to a higher meat color stability in NR-supplemented pork during retail display.

Radiofrequency Induced Time-Dependent Alterations in Gene Expression and Apoptosis in Glioblastoma Cell Line.

The widespread use of wireless communication technologies has increased human exposure to radiofrequency electromagnetic fields (RF-EMFs). Considering the brain's close proximity to mobile phones and its entirely electrical transmission network, it emerges as the organ most profoundly impacted by the RF field. This study aims to investigate the potential effects of RF radiation on cell viability, apoptosis, and gene expressions in glioblastoma cells (U118-MG) at different exposure times (1, 24, and 48 h). To achieve this, we designed and implemented an in vitro RF exposure system operating at a frequency of 2.1 GHz, specifically for cell culture studies, with an average specific absorption rate (SAR) of 1.12 ± 0.18 W/kg determined through numerical dosimetry calculations. Results reveal a significant influence of a 48 h exposure to a 2.1 GHz RF field on U118-MG cell viability, gene expression, and the induction of caspase (CASP) dependent apoptosis. Notably, increased CASP3, CASP8, and CASP9 mRNA levels were observed after 24 and 48 h of RF treatment. However, only the 48 h RF exposure resulted in apoptotic cell death and a significant elevation in the BAX/BCL-2 ratio. This observed effect may be influenced by extended exposure durations surpassing the cell's doubling time. The increased BAX/BCL-2 ratio, which acts as a key switch for apoptosis, and the heterogeneous morphology of the astrocyte-derived U118-MG cell line may also play a role in this effect.

The Role of Flavonoids from Aurantii Fructus Immaturus in the Alleviation of Allergic Asthma: Theoretical and Practical Insights.

Flavonoids derived from plants in the citrus family can have an alleviating effect on allergic asthma. The aim of this study was to provide insights into the mechanisms by which these compounds exert their effects on allergic asthma by combining theoretical and practical approaches. Aurantii Fructus Immaturus flavonoids (AFIFs) were obtained by solvent extraction and were determined by high performance liquid chromatography (HPLC). In vivo and in vitro experiments combined with network pharmacology, Mendelian randomization (MR) analysis and the AutoDock method were applied to study the mechanism of their effects. The main AFIFs were found to be hesperidin (13.21 mg/g), neohesperidin (287.26 mg/g), naringin (322.56 mg/g), and narirutin (19.35 mg/g). Based on the network pharmacology and MR analysis results, five targets Caspase 3 (CASP3), CyclinD1 (CCND1), Intercellular adhesion molecule (ICAM), erb-b2 receptor tyrosine kinase 2 (ERBB2), and rubisco accumulation factor 1 (RAF1) were selected, and the interactions between the AFIFs and the targets were studied using AutoDock Vina. The results indicated that glycosidic bonds play an important role in the interactions between AFIFs and both ERBB2 and RAF1.

Assessing hepatotoxicity induced by co-exposure to chlorpyrifos and deltamethrin in hook snout carp (Opsariichthys bidens Günther): A comprehensive analysis biochemical and molecular response analysis.

Chlorpyrifos (CLP) and deltamethrin (DTM) are among the most widely utilized organophosphate and pyrethroid insecticides globally. Their simultaneous presence in aquatic environments poses significant threats to fish health and challenges the sustainability of aquaculture practices. Despite their prevalence, the combined toxic effects of CLP and DTM on hook snout carp (Opsariichthys bidens Günther) remain insufficiently understood. In this study, O. bidens were exposed to waterborne treatments of CLP, DTM, or their combination for 30 days, and the biochemical and molecular responses of the liver tissue were systematically assessed. Acute toxicity tests revealed that the combined exposure to CLP and DTM resulted in synergistic toxicity. Significant alterations in the activities of key enzymes, including superoxide dismutase (SOD), catalase (CAT), caspase-3 (CASP-3), and caspase-9 (CASP-9), relative to the control group, demonstrated that co-exposure induced oxidative stress in O. bidens. Additionally, the elevated transcriptional levels of immune-related genes such as cxcl-c1c, il-8, and il-1 suggested a pronounced inflammatory response triggered by the pesticide mixture. Conversely, the significantly reduced expression of p53 and esr indicated that combined exposure disrupted apoptotic regulation and endocrine system function in the fish. In summary, these findings demonstrated that co-exposure to CLP and DTM induced liver damage in O. bidens by impairing antioxidant enzyme activity, disrupting apoptosis regulation, and altering the transcriptional profiles of genes involved in immune and endocrine pathways. These results provided new insights into the physiological and molecular mechanisms of pesticide-induced hepatotoxicity in fish and offered valuable information for evaluating the ecological risks associated with pesticide mixtures in aquatic environments.

Integrated application of target-based and ligand-based drug-designing approaches for the identification of novel caspase-6 inhibitors

Caspase-6 (CASP6) is an effector caspase that has been marked to possess various pathological attributes associated with neurodegeneration. It is widely expressed in the neurodegenerative brain and peripheral tissues. It plays a vital role in apoptotic cell death and also performs non-apoptotic functions like axon pruning which contribute to the degeneration of neurons. Increment in active CASP6 levels in the cerebrospinal fluid has been observed during inflammation and has been linked to the early onset of Alzheimer’s disease (AD). In the current study, a novel CASP6 inhibitor was identified with the help of integrated target-based and ligand-based drug-designing approaches. Various molecular features of US9 (PDB ID 8EG6) were used to generate models. The pharmacophore models were evaluated using the EF value, GH score, and percentage yield to select the best-suited model. The best model was used to screen the ZINC-15 database to obtain virtual hits. The undesirable compounds were eliminated using various nodes in KNIME workflow. The resulting compounds were further subjected to docking-based virtual screening (DBVS) to find the lead compounds. Further, the molecular docking studies were carried out in three stages, followed by pharmacokinetic property prediction and toxicity studies. The top two virtual hits, i.e. ZINC000012563650 and ZINC000069415222, were considered for molecular dynamics simulation studies. Compound ZINC000069415222 was found to possess better stability, drug-like properties, and lower toxicity under simulated conditions. Thus, ZINC000069415222 was identified as a potential CASP6 inhibitor that could be further explored experimentally as an anti-AD drug.

Exploring the therapeutic potential of puerarin on intervertebral disc degeneration by regulating apoptosis of nucleus pulposus cells.

Intervertebral disc degeneration (IVDD) stands as a prevalent chronic orthopedic ailment, profoundly impacting patients' well-being due to incapacitating low back pain. Studies have highlighted a close correlation between IVDD and the programmed cell death of nucleus pulposus (NP) cells orchestrated by interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and caspase-3 (CASP3). Puerarin, renowned for its anti-inflammatory attributes and its influence on IL-1β and TNF-α, emerges as a promising candidate for IVDD treatment. However, the precise mechanism by which it regulates apoptosis via these pathways remains ambiguous. This investigation utilizes bioinformatics to unveil the molecular intricacies of puerarin-mediated apoptosis regulation in IVDD, substantiated by preliminary in vitro experiments. Analysis exposes aberrant expression of pivotal apoptosis-associated proteins (IL-1β, TNF-α, CASP3, CASP8, and BCL2) in IVDD patients, with network pharmacology indicating puerarin's potential efficacy in IVDD treatment by modulating apoptosis and cellular senescence pathways. Further experiments elucidate puerarin's capacity to stimulate NP cell proliferation while inhibiting apoptosis, potentially contributing to IVDD mitigation. Western blot and PCR outcomes reveal escalated expression of apoptosis-related proteins (IL-1β, TNF-α, and CASP3) in lipopolysaccharide-treated NPCs, ameliorated by puerarin intervention. Molecular docking simulations demonstrate favorable binding properties of puerarin with apoptotic proteins, while flow cytometry analysis indicates its ability to diminish NPC apoptosis. These discoveries imply that puerarin might alleviate NPC apoptosis by modulating key targets, thereby potentially ameliorating IVDD. In summary, this study unveils the intrinsic mechanism of puerarin in regulating NPC apoptosis to alleviate IVDD, underscoring its therapeutic promise.

Network Pharmacology and Bioinformatics Analyses Identify the Core Genes and Pyroptosis-Related Mechanisms of Nardostachys Chinensis for Atrial Fibrillation.

Nardostachys chinensis is an herbal medicine widely used in the treatment of atrial fibrillation (AF), but the mechanism is unclear. To explore the molecular mechanism of N. chinensis against AF. The TCMSP was used to screen the active N. chinensis compounds and their targets. Differentially expressed genes (DEGs) for AF were identified using open-access databases. Using Venn diagrams, the cross-targets of N. chinensis, pyroptosis, and AF were obtained. The genes underwent molecular docking as well as gene set enrichment analysis (GSEA). A nomogram based on candidate genes was constructed and evaluated with the clinical impact curve. After that, the immune infiltration of the dataset was analyzed by single sample GSEA (ssGSEA). Finally, microRNAs (miRNAs) and transcription factors (TFs) were predicted based on candidate genes. Tumor necrosis factor (TNF) and caspase-8 (CASP8) were obtained as candidate genes by taking the intersection of DEGs, targets of N. chinensis, and pyroptosis-related genes. Tolllike receptor (TLR) and peroxisome proliferator-activated receptor (PPAR) signaling pathways were linked to candidate genes. Additionally, immune cell infiltration analysis revealed that CASP8 was associated with natural killer T cells, natural killer cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), macrophages, CD8 T cells, and CD4 T cells. Finally, miR-34a-5p and several TFs were found to regulate the expression of CASP8 and TNF. CASP8 and TNF are potential targets of N. chinensis intervention in pyroptosisrelated AF, and the TLR/NLRP3 signaling pathway may be associated with this process.

2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway.

To investigate the anti-liver cancer effect of 2-hydroxy-3-methyl anthraquinone (HMA) and the specific mechanism based on nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1 (SIRT1)/cellular tumor antigen p53 (p53) pathway. Cell counting kit-8 method was used to observe the effect of HMA on the activity of human hepatocellular carcinoma cells (HepG2) cells. At 72 h and 80 μL HMA, the apoptosis rate of HepG2 cells in each group was measured by flow cytometry. Transwell was used to assay for cell invasion. The protein expression levels of SIRT1, p53, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-9 (CASP9) and caspase-3 (CASP3) were detected by Western Blot. HMA significantly inhibited the proliferation of HepG2 cells, The half inhibiting concentration (IC50) of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively. Compared with the control group, the apoptosis rate of HMA, Selisistat (EX527), and HMA+ EX527 groups enhanced, while the apoptosis rate of SRT1720 diminished, demonstrating that inhibition of SIRT1 can lead to apoptosis of HepG2 cells. HMA+ EX527 group had the highest apoptosis rate, the lowest expression of SIRT1 and Bcl-2, and the highest expression of p53, Bax, CASP9 and CASP3. The number of invasions of HepG2 was significantly reduced after HMA and EX527 intervened. Western blot shows HMA could inhibit SIRT1, promote the expression of p53, and decrease the ratio of Bcl-2/Bax. HMA induced apoptosis in HepG2 cells, while inhibiting proliferation and invasion. The mechanism of HMA against HCC may be related to the SIRT1/p53 pathway.

Kaempferol alleviates rheumatoid arthritis through pyroptosis based on bioinformatics analysis and experimental validation

Kaempferol has an anti-inflammatory effect on rheumatoid arthritis (RA), but the mechanism by which inflammatory reactions are relieved via pyroptosis is still unclear. In the present study, The bioinformatics technology was used to analyze the relationship between pyroptosis the hub genes and the downstream cytokines in RA from three the Gene Expression Omnibus (GSE12021, GSE1919 and GSE29746). Molecular docking analysis of kaempferol and caspase1 (CASP1) was executed with Autodock tools. RA fibroblast-like synoviocytes (RA-FLS) was used to explore the anti-inflammatory effects of kaempferol on RA via pyroptosis. Thirty-seven differentially expressed genes (DEGs) were identified from the three datasets, and the enrichment analysis was focused mainly on the chemokine signaling pathway. The difference in immune cell infiltration was focused on CD4 T cells, CD8 T cells, M1 macrophages, and M2 macrophages. The expression of CASP1 and caspase3 (CASP3) was higher in the RA group. CASP1 and CASP3 were positively correlated with cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Moreover, molecular docking revealed that kaempferol strongly interacts with CASP1. Kaempferol inhibited the proliferation and migration of RA-FLS in vitro. The expression of CASP1, CASP3, caspase5 (CASP4), caspase5 (CASP5), gasdermin D (GSDMD), N-terminal domain of gasdermin D (GSDMDNT), IL-1β, and cytokines interleukin-18 (IL-18) were significantly affected by kaempferol. Kaempferol alleviated RA-FLS membrane disruption, and it blocked CASP1 to alleviate RA via pyroptosis. The present study demonstrated that kaempferol binds to CASP1 and affects pyroptosis in RA, thereby serving as an anti-immunotherapy strategy.

Novel Variants in Medium and Low Penetrance Predisposing Genes in a Hungarian Malignant Melanoma Cohort With Increased Risk.

Both germline and somatic variants contribute to the genetic background and pathogenesis of melanoma. Germline variants include the presence of rare pathogenic or likely pathogenic variants of high, medium, and low penetrance melanoma-predisposing genes. Rare variants of high penetrance melanoma-predisposing genes are associated with melanoma development, whereas the medium and low penetrance predisposing genes can significantly increase melanoma risk. In this study, we clarified the germline genetic background of a Hungarian melanoma cohort (n = 17). Using a gene panel of 30 melanoma-predisposing genes, germline genetic variants were identified in 10 of the 17 patients (58.82%). A novel, likely pathogenic, missense variant (p.Y143C) in a medium penetrance melanoma-predisposing gene, melanocortin 1 receptor gene (MC1R), and two novel, likely pathogenic nonsense variants in low penetrance genes, p.Q218Ter in caspase 8 (CASP8) and p.Q40Ter in the fat mass- and obesity-associated (FTO) gene were detected. This study highlights the importance of elucidating the germline genetic background of melanoma, which may improve prediction of individual risk and the risk of family members and to optimize preventive, screening, and therapeutic measures for each patient and melanoma-prone families.

Association of CASP8 rs3834129 and CTGF rs6918698 genotypes with susceptibility to colorectal cancer in a Mexican population.

Connective tissue growth factor (CTGF) and Caspase 8 (CASP8) have been implicated in cancer development and progression. Variants such as CASP8 rs3834129 (-652 6N I/D) and CTGF rs6918698 (-945 C>G) have been associated with several cancers, although their association is still debated between populations. This study investigates the possible association between the CASP8 rs3834129 and CTGF rs6918698 variants with colorectal cancer (CRC) in Mexican patients. Genomic DNA was extracted from 250 CRC patients and 250 control subjects. The identification of CASP8 and CTGF variants was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. The association was determined by the odds ratio (OR) analysis and P values were adjusted by the Bonferroni correction. Patients carrying the D/D genotype for the CASP8 rs3834129 variant exhibited an increased susceptibility to CRC (P = 0.012). The D/D genotype was associated with older 50-year-old patients (P = 0.006). In addition, this same D/D genotype was associated with TNM II stage (P = 0.013) and rectal localization (P = 0.023). Additionally, patients carrying the G/G genotype for the CTGF rs6918698 variant showed a decreased susceptibility to CRC (P = 0.009), and in the sex stratification, this gene has protective role in males (P = 0.008). This same genotype was associated with decreased susceptibility to early TNM stages (I+II) (P = 0.023) and right-sided colon tumor localization (P = 0.002). There was no association between response to treatment and the variants analyzed. Our findings suggest that the CASP8 rs3834129 and CTGF rs6918698 variants have a significant impact on the development of CRC.

Comparative evaluation of the modulatory role of 1,25-dihydroxy-vitamin D3 on endoplasmic reticulum stress-induced effects in 2D and 3D cultures of the intestinal porcine epithelial cell line IPEC-J2

BackgroundThe use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells.ResultsA published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture.ConclusionsThe effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model.

SYNERGISTIC POTENTIAL OF NIGELLA SATIVA L. AND TRIGONELLA FOENUM-GRAECUM: INTEGRATED NETWORK PHARMACOLOGY FOR DIABETIC WOUND HEALING

Objective: Diabetes Mellitus (DM) is a metabolic disorder marked by elevated blood glucose levels, and one of the issues linked to DM involves the development of Diabetic Wounds (DW). DW is susceptible to infection and develops into chronic wounds if not treated properly. This study aimed to investigate the network pharmacology of N. sativa L. and T. foenum-graecum, emphasizing on their potential as DW treatment candidates. Methods: Various databases were used in this study, including PubChem, Dr. Duke's phytochemistry and Ethnobotany, and KNApSAcK Family. Swiss Target Prediction and Way2Drug PASS Online were utilized for biological activity and protein target prediction. The DW pathway's protein-protein interactions were examined with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Cards, and STRING databases. STRING was used to predict the metabolite's action. The relationship between metabolites and target proteins was predicted using STITCH, and Cytoscape was used to visualize the network. Result: The results showed that ten active ingredients (five active ingredients in N. sativa L. and five active ingredients in T. foenoem-graecum) contributed to DW healing by affecting Tumor Necrosis Factor (TNF), Interleukin-1beta (IL1B), JUN, Caspase 3 (CASP3), Interleukin-6 (IL-6), Alpha Kinase Threonine-1 (AKT1), Vascular Endothelial Growth Factor-A (VEGFA), and Mitogen-Activated Protein Kinase 3 (MAPK3) genes. Furthermore, the ten active ingredients correlated with twenty-eight intracellular proteins, resulting in a mechanism involving eight DW signalling pathways. Conclusion: Based on network pharmacology analysis, we determine that N. sativa L. and T. foenoem-graecum combination can potentially treat DW.

The Impact of Perfluorooctanoic Acid (PFOA) on the Mussel Mytilus galloprovincialis: A Multi-Biomarker Evaluation

Perfluorooctanoic acid (PFOA) has been widely studied due to its environmental persistence and bioaccumulation potential, raising concerns about its effects on aquatic life. This research evaluates the impact of PFOA on the antioxidant defenses and stress response systems of the mussel Mytilus galloprovincialis. Mussels were exposed to three concentrations of PFOA (1, 10, and 100 µg·L−1) over 28 days. Several biomarkers, including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO), total antioxidant capacity (TAC), vitellogenin (VTG), ubiquitin (UBI), and caspase-3 (CASP) were analyzed. The results suggest stress responses, particularly in animals exposed to higher concentrations, as shown by GST and SOD activities which increased according to PFOA concentrations. Additionally, oxidative stress markers such as MDA and CAT showed variable responses depending on the exposure concentration tested. This study underscores the need for further investigation into the effects of PFOA on mollusks but also the need to unveil gender-specific responses in aquatic organisms exposed to this contaminant. The concentrations of PFOA used in our research are lower than those examined in previous studies, providing crucial insights into the impacts of even minimal exposure levels. It highlights the potential of M. galloprovincialis as a bioindicator in environmental monitoring programs, providing crucial insights for environmental management and policymaking regarding regulating and monitoring PFOA in marine settings. Consequently, in a country where seafood consumption is the second largest in Europe, implementing environmental policies and regulatory measures to manage and monitor PFOA levels in marine environments is crucial.

Paralichthys olivaceus GSDME-mediated pyroptosis is regulated by multiple caspases in different manners

Pyroptosis is a type of programmed cell death mediated by gasdermin (GSDM). GSDM is activated by caspase (CASP), which cleaves GSDM to release the N-terminal (NT) fragment that forms channels in the plasma membrane and leads to cell death. To date, research on pyroptosis in teleost is limited. In this study, we examined the activation and regulation mechanism of pyroptosis in flounder Paralichthys olivaceus. P. olivaceus gasdermin E (PoGSDME) was found to be cleaved by six P. olivaceus caspases (PoCASP1/3a/3b/7/8a/8b). PoCASP1/3a/3b/7 cleaved primarily at 245FEAD248, which generated an NT fragment (NT248) that induced robust pyroptosis. PoCASP8a/8b cleaved both the full length PoGSDME and NT248 at 202IEKD205, thus destroying the biological activity of PoGSDME and NT248. Nine residues crucial for PoGSDME function were identified, of which, F2, L19, and G85 were essential to plasma membrane translocation. During bacterial infection, PoGSDME and PoCASP1 expressions were significantly upregulated in flounder tissues, and PoGSDME, as well as PoCASP1, activation occurred in flounder cells accompanied with the processing cleavage of IL-1β and IL-18. Together these results revealed both the activation and the inhibition mechanisms of GSDME-mediated pyroptosis in flounder, and added new insights into the regulation of pyroptosis in fish.

Antiaging properties of chlorogenic acid through protein and gene biomarkers in human skin fibroblast cells as photoaging model

Background and purpose: Chlorogenic acid (CA) is a natural chemical that promises antiaging activity against photoaging skin damage. This research examined CA activities in mitigating skin photoaging. Experimental approach: UV-exposed human skin fibroblast cells were subjected to CA at 6.25, 12.5, and 25 μg/mL. The protein levels of cell secretion, such as cyclooxygenase (COX)-2, nitric oxide (NO), and interleukin (IL)-6 were measured using ELISA and colorimetry methods. Meanwhile, the mRNA expressions of glutathione peroxidase (GPX)-1, tissue inhibitor metalloproteinase (TIMP)-1, matrix metalloproteinase (MMP)-1, caspase (CASP)-3, CASP-8, and fibroblast growth factor (FGF)-2 were quantified using the qRT-PCR method. Findings/Results: CA treatment reduced inflammatory and aging biomarkers. CA at 6.25 μg/mL lowered NO, COX-2, and IL-6 levels to 89.44 μmol/L, 8.10 ng/mL, and 62.75 pg/mL, respectively. CA at 25 μg/mL resulted in the most significant down-regulation of MMP-1, CASP-3, and CASP-8 genes’ expression (3.27, 1.25, and 3.59, respectively). Furthermore, treatment with CA at 25 µg/mL demonstrated the most notable activity in up-regulating antioxidant markers, specifically GPX-1, and extracellular matrix (ECM) integrity markers, including TIMP-1 and FGF-2 genes’ expression. Conclusion: CA imposes its anti-aging activity by decreasing inflammatory and aging biomarkers, and increasing cellular antioxidant and ECM integrity.

Mechanism of osthole against colorectal cancer based on network pharmacology, molecular docking, and experimental validation

Through in vitro and in vivo experiments, combined with network pharmacology and molecular docking techniques, this study investigated the mechanism of action of osthole in the treatment of colorectal cancer(CRC). The relevant targets of osthole and CRC were retrieved from the SwissTargetPrediction and SuperPred in drug databases, as well as GeneCards and OMIM in disease databases. Protein-protein interaction(PPI) networks were constructed using the STRING database and Cytoscape 3.8.0 software, and core targets were screened. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed on common targets. Molecular docking validation of core targets with osthole was conducted using AutoDock Vina software. HCT116 cells were treated with different concentrations of osthole, and cell proliferation was detected using the CCK-8 assay and the clonogenic assay. Cell migration ability was assessed using Transwell assay. Western blot and RT-qPCR were performed to detect the expression of caspase-3(CASP3), hypoxia-inducible factor 1 alpha(HIF1A), nuclear factor kappa B subunit 1(NFKB1), glycogen synthase kinase-3 beta(GSK3B), phosphorylated-GSK3B(p-GSK3B), protein kinase B(Akt), phosphorylated-Akt(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated-mTOR(p-mTOR). A subcutaneous tumor model of HCT116 cells in nude mice was established, and the mice were randomly divided into the model group, low-dose osthole group(20 mg·kg~(-1)), medium-dose osthole group(40 mg·kg~(-1)), and high-dose osthole group(60 mg·kg~(-1)). After 18 days of administration, the growth of tumor xenografts was observed, and the size and weight of tumors were measured after excision. Hematoxylin-eosin(HE) staining was performed to observe the histological changes in tumors in each group. Network pharmacology analysis revealed that osthole treatment of CRC mainly involved 106 treatment targets and 113 treatment pathways, with key pathways including the PI3K/Akt signaling pathway and MAPK signaling pathway. Molecular docking results showed a strong correlation between osthole and core targets. In vitro studies demonstrated that osthole significantly inhibited the proliferation and migration ability of HCT116 cells. Western blot and RT-qPCR experiments showed that compared to those in the model group, the expression of NFKB1, HIF1A, p-Akt, p-mTOR, and GSK3B in the osthole-treated group was significantly decreased, while the expression of CASP3 and p-GSK3B(Ser9) was significantly increased. In vivo studies showed that compared to the model group, osthole-fed animals significantly reduced tumor weight and volume, inhibited tumor growth, and promoted tumor apoptosis, and the results showed a dose-dependent trend. The study suggested that osthole could inhibit the proliferation and migration of HCT116 cells in CRC, and its mechanism may be related to the regulation of the PI3K/Akt signaling pathway and the expression of core targets.

Caspase-2 is a condensate-mediated deubiquitinase in protein quality control

Protein ubiquitination plays a critical role in protein quality control in response to cellular stress. The excessive accumulation of ubiquitinated conjugates can be detrimental to cells and is recognized as a hallmark of multiple neurodegenerative diseases. However, an in-depth understanding of how the excessive ubiquitin chains are removed to maintain ubiquitin homeostasis post stress remains largely unclear. Here we found that caspase-2 (CASP2) accumulates in a ubiquitin and proteasome-positive biomolecular condensate, which we named ubstressome, following stress and functions as a deubiquitinase to remove overloaded ubiquitin chains on proteins prone to misfolding. Mechanistically, CASP2 binds to the poly-ubiquitinated conjugates through its allosteric ubiquitin-interacting motif-like region and decreases overloaded ubiquitin chains in a protease-dependent manner to promote substrate degradation. CASP2 deficiency in mice results in excessive accumulation of poly-ubiquitinated TAR DNA-binding protein 43, leading to motor defects. Our findings uncover a stress-evoked deubiquitinating activity of CASP2 in the maintenance of cellular ubiquitin homeostasis, which differs from the well-known roles of caspase in apoptosis and inflammation. These data also reveal unrecognized protein quality control functions of condensates in the removal of stress-induced ubiquitin chains.

Role of layilin in regulating mitochondria-mediated apoptosis: a study on B cell lymphoma (BCL)-2 family proteins

BackgroundMalignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells.ResultsWe compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions.ConclusionThis study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis.